Choosing the best fit cell culture harvesting technology for your process
Posted on September 27, 2018
The development of the “Mammalian Cell Culture Harvesting Guide”
Silke Bergheim, Global Product Manager, Depth Filtration
The development of the “Mammalian Cell Culture Harvesting Guide”
Which cell culture harvesting technology to use in a monoclonal antibody (mAb) process is decided early in process development. Each cell culture has unique characteristics and the Mammalian Cell Culture Harvesting Guide has been developed to enable process developers to make the right decision at this early stage. The Guide describes the three most important cell culture harvesting platforms and explains the criteria to choose the best fit technology to get the strongest performing harvest process. In this article the three technologies are compared on a technical, ease of handling and cost basis.
In the last ten years manufacturing of monoclonal antibodies has become a commercial reality. Continuous optimization has improved the productivity of the cells and high cell titers and mAb concentrations are standard today, but this improvement has been accompanied by the generation of more biomass and impurities and therefore the harvesting process is a bigger challenge than it was 10 years ago.
Modern processes with high cell titers and mAb concentrations are often manufactured at smaller scales than in the past, up to approximately 2,000L and use single-use components in manufacture. The use of a centrifuge for cell harvesting, a capital intensive step, is replaced by modern single- use harvesting platforms.
The three most attractive and popular alternatives to a centrifuge are: single-use depth filtration, acoustic separation, and the body feed method with diatomaceous earth. For this article these three technologies are compared on a technical and costs basis.
Single-Use Depth Filtration
For single-use depth filtration, the Pall Stax™ mAx filter was evaluated. The primary clarification step is performed by a double layer of PDP8 filter sheet with a retention rating of 30-6µm. To achieve a turbidity of less than 10 NTU, a secondary clarification step using a PDE2 double layer filter sheet with a retention rate of 3.5-0.2 µm was used.
The acoustic separation was performed with the Pall Cadence™ Acoustic Separator.
The technology involves the use of low frequency acoustic forces to generate a 3-dimensional (3D) standing wave across a flow channel. Cell culture from a fed batch bioreactor enters the flow channel, and as the cells pass through the 3D standing wave they are trapped by the acoustic forces. The trapped cells migrate to the nodes of the standing wave, and begin to clump together until such time as their buoyancy decreases and they settle out of the suspension by gravity. The residual particles were further clarified by depth filtration. Compared with using just depth filtration the required filter area was reduced by 80%.
The final turbidity was less than 10 NTU.
Body Feed Method
To perform the body feed method, diatomacious earth was added to a Pall LevMixer® containing the cell culture using a Pall powder handling bag. The solution was filtered through Pall Stax CF cake filtration capsules which provide enough space to allow a diatomacious earth filter cake to build up, this clarifies the cell culture to a final turbidity of less than 10 NTU.
For the study 25 different cell cultures standardized on a 1000L cell culture formulation were harvested with all three technologies. The results were evaluated for ease of handling, meeting critical parameters and cost.
Ease of Use and Footprint
In comparison, single-use depth filtration is the simplest technology to use and requires the smallest footprint. For larger processes requiring more than 20 capsules and two chassis, this may not be the most economic solution. The first step is to install the manifold plate and the capsules, connect both and rinse the system. After filtration and blow down, the capsules and manifold plates are dismantled and disposed of.
The 1000L process scale Cadence Acoustic Separator has a larger footprint and additional space is required for the secondary depth filter system, although this is approximately 80% smaller than when depth filtration alone is used for the clarification processing step. The acoustic separator manifold and the depth filter manifold plate and capsules are installed. Both are rinsed before harvesting and after filtration dismantled and disposed of. The complexity of the operation is comparable to that of depth filtration alone.
The body feed method requires more space and work input compared to the other technologies. For particle free addition the diatomaceous earth needs to be supplied in powder-handling-bags. The bag is connected to a single-use mixer unit and the diatomaceous earth powder is released into the cell culture and mixed in. To prepare the process step a single-use mixer bag and the required number of depth filters and manifold plates are installed. The units are connected with a single-use manifold, rinsed before use and blown down, dismantled and disposed of after use. For a 1000L cell culture, several diatomaceous earth portions of manageable size are added and mixed in, which causes a high work load and increases the process time.
For the highest mAb yield the mammalian cell culture is optimized on cell line, clone, cell culture media and process train. The most relevant process control parameters are: cell density [cells/ml], turbidity [NTU] and pack cell mass [PCM %].
For the development of the Mammalian Cell Culture Harvesting Guide these process control parameters were defined as the most critical ones and the influence on the different technologies were determined.
25 cell cultures with a cell density variance of 6 to 60 x106 cells/ml, a turbidity of 800 to 4000 NTU and a pack cell mass of 2 to 30% were harvested with the three technologies and the results were compared.
Cost Benefit Analysis
For cell densities up to around 35 x106 cells/ml and a turbidity of 3000 NTU, the single-use depth filters performed well. Cell cultures with higher cell counts and turbidities required an increased filtration area, which was not economic compared to the other technologies.
From a technical point of view, the acoustic separation showed a consistently good performance through all cell densities and turbidity. To stay within a harvesting time of around 5-6 hours more separation chambers and also higher filter areas were necessary with increasing cell densities. To operate a higher number of chambers, hardware modules need to be installed in addition to the basic unit. Even though this means a higher investment and more consumables the technology stays economic for cell culture cell densities of 35 x106 cells/ml and higher.
The body feed method with cake filtration is very robust against increasing cell densities and turbidities, but it strongly depends on the pack cell mass. The higher the PCM the more diatomaceous earth must be added to bind the cells, cell debris and impurities and effectively form a filtration cake. The ratio of PCM to diatomaceous earth is 1:2 for a cell culture with pH=7 and 1:4 for a pH-reduced cell culture or PH= 5. With increased pack cell mass, the number of cake filtration capsules needed increases, as does the work load for adding the diatomaceous earth and the time required to mix it into the cell culture. Increased powder additions and mixing times also increase the harvest time. The body feed method is economic for pack cell masses up to 15-20% which are normal with cell densities of less than 35 x106 cells/ml. In consequence of the high work load the method is too time consuming for cell cultures of more than 1000L as the harvesting time would be more than 8 hours.
Summary: The Mammalian Cell Culture Harvesting Guide
In regard to economics, the comparison of the three harvesting technologies determined on a data basis of harvesting 25 cell cultures with different cell densities, turbidities and pack cell mass, indicates that the depth filtration and body feed method are both preferable for cell cultures with cell densities up to 35 x106 cells/ml. For higher cell densities acoustic separation is more economic.
Taking cell density as the first relevant criteria, the turbidity of the cell culture gives an indication of which technology would perform best when the cell density is less than 35 x106 cells/ml. Depth filtration is most economic up to a turbidity of 3000 NTU. Other advantages of this method are the small space requirement and simple handling. Above a turbidity of 3000 NTU the body feed method is the most economic one but it must be considered that harvesting times can be long and work intensive for bigger cell culture scales and high pack cell mass.
Above a cell density of 35 x106 cells/ml the body feed method can still be attractive for small scale cell cultures and up to pack cell masses of 15-20%. Economically more interesting for high cell densities up to 10 x106 cells/ml and for perfusion processes is acoustic separation.
The Mammalian Cell Culture Harvesting Guide is based on the results of tests completed on 25 different cell cultures. For a quick orientation, the guide shows the evaluation described above as a decision tree and supports process development scientists to find the best fit cell harvest technology for their process.
It can be downloaded from http://go.pall.com/Stax-mAx-clarification-new.html
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